Project Summary. Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients, particularly those with acute leukemia, hematopoietic cell transplantation, and recipients of chronic corticosteroid therapy for graft versus host disease and autoimmunity. Despite recent advances in antifungal therapies, it remains poorly understood which fungal and host factors are critical for disease progression after establishment of infection in the lung. We have made 2 fundamental observations toward narrowing this knowledge gap. First, the causative agent of IA, Aspergillus fumigatus, interacts with host tissue to generate a dynamic oxygen microenvironment at the site of infection. Second, A. fumigatus adapts to established infection microenvironments by exhibiting infection site-specific metabolic flexibility that is critical for fungal virulence. We recently termed these adaptations ?disease progression factors? (DPFs) because they are essential for the progression of invasive disease and relatively undefined in the context of human fungal infections. An exciting recent discovery is the previously unappreciated role of the fungal oxygen response genetic network, which includes the DPFs, SrbA, SrbB and CreA, in responses to transitions in oxygen tension. Our recent data lead us to hypothesize that this oxygen response network regulates the production of specific metabolites that promote and support fungal disease progression. A major effector of this genetic network, an unstudied fungal alanine aminotransferase alaA, is the focus of mechanistic studies in Aim 1 of this proposal. We propose a model whereby alaA functions as key regulator of metabolic transitions required for adaptation to oxygen fluctuations during fungal disease progression. In aim 2, we build off a novel genetic screen which has identified four new unstudied fungal transcription factors that are critical for the response to oxygen tension fluctuations. We have named these new genes ortA-D for oxygen responsive fungal transcription factors. An innovation to our approach to test our hypotheses and models is the incorporation of in vivo imaging of the infection site microenvironment during disease progression that is revealing new insights into fungal form and function in an established infection. Consequently, at the conclusion of these studies, we will have defined new molecular mechanisms of fungal fitness in established infection environments that are expected to reveal new therapeutic opportunities to improve disease outcomes for these too often lethal invasive mold infections.